ABSTRACT Reaching a more complete understanding of biological processes and mechanisms that underlie health and disease demands a better integration of information spanning multiple length and time scales. Super-resolution microscopy and single-molecule approaches have emerged as potent tools that extend the spatial resolution and detection sensitivity in live biological imaging. However, the current state-of-the-art techniques often achieve limited 3D resolution that precludes visualizing spatial organization at the molecular scale. Moreover balancing trade-offs between temporal and spatial resolution, while operating with a limited photon budget often results in severely shortened single-molecule observation times. Finally, many microscope configurations are challenged when imaging weak signals from single- molecules, especially due to high background in crowded cellular specimens. Thus, although promising, the full potential of single-molecule/super-resolution methods for transforming our molecular understanding of biological processes has yet to be realized. To fill critical technical gaps, new optimized microscope configurations are needed - that can operate at the limits of spatiotemporal resolution while maximizing the information content of dim fluorescence signals. We hypothesize that this goal can be achieved through novel combinations of 3D interferometry, targeted fluorescence switching, while further harnessing emerging photon-efficient algorithms to increase resolution as well as prolong total observation times. Based on these ideas we propose to develop novel super-resolution and single-molecule fluorescence imaging tools, focusing on two specific aims: (1) To extend the spatiotemporal scales of localization-based single-molecule imaging and tracking to 1 nanometer isotropic 3D resolution and to ~1,000 data-point in vivo observation traces at down to (sub)millisecond sampling rates; (2) To achieve real-time single-molecule detection sensitivity in addressable 3D volumes, at presence of micro- Molar background concentrations, and inside highly crowded intracellular environments. The new techniques will significantly increase our abilities to interrogate dynamic biological processes with molecular detail, thus having widespread and immediate impact across biomedical disciplines.